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. 2020 May 8;3:225. doi: 10.1038/s42003-020-0960-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Example traces of PER2::LUC expression over 7 days in culture at the whole tissue level in the AP, NTS and 4^thVep from one brainstem slice. b Cross-correlograms showing the ~2 h lag of NTS to AP (left panel; n = 8) and close to antiphasic relationship of 4^thVep to AP (right panel; n = 5) over 72 h. c Altered times of peak PER2::LUC bioluminescence (AP vs NTS ****p < 0.0001, n = 8, t test; 4^thVep vs AP and NTS ^###p < 0.001, n = 5, Tukey’s test). d Periods of whole area structures of the brainstem (^#p < 0.05, n = 5, Tukey’s test). e Reconstructed images from grid-based, normalised PER2::LUC activities in the brainstem slice showing phase of spatial PER2::LUC expression every 4 h over 3 days. Warm colours code toward peak phase. f (from left to right) The average phase of the oscillation for the first 24 h in culture (upper left) and the period for the first 48 h (lower left), the map of clustering based on the correlation of time series (middle), and the raster plot of normalised time series of the oscillation listed in cluster groups (each row represents one grid) (right). The first cluster (red) approximates the anatomical position of NTS, the second one (green) the 4^thVep, and the third cluster (blue) the AP (middle). g Traces of putative single cell PER2::LUC expression over 7 days in culture. h Rayleigh plots depicting phase clustering around CT 8.5 in AP and CT 11 in NTS at day 2, and subsequent desynchronisation of single cell oscillators at day 5. i Rayleigh r value of individual cells for peaks 2-5 (left panel) and the SD of individual cell lags relative to the phase of the main structure for peaks 2-5 (right panel) for n = 8 cultures. (****p < 0.0001, two-way ANOVA RM). J. Frequency histogram of single cell lags relative to the phase of the whole structure (****p < 0.0001, two-way ANOVA Interaction). k In vivo core clock gene expression of Per2 and Bmal1 in the AP and NTS relative to CT0. (*p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA).